1. Field of the Invention
The field of this invention is hybridomas and monoclonal antibodies. More specifically, this invention relates to hybridoma produced monoclonal antibodies that can specifically identify endothelial cells in diseased tissue, and which are useful in the diagnosis of rheumatoid and osteoarthritis synovial tissues and psoriatic skin.
2. Description of the Prior Art
The fusion of mouse myeloma cells and spleen cells from immunized mice by Kohler and Milstein in 1975 (Nature 256:495-497, 1975) demonstrated for the first time that it was possible to obtain a continuous cell line making homogeneous (so-called "monoclonal") antibody. Since this seminal work, much effort has been directed to the production of various hybrid cells (called "hybridomas") and to the use of the antibody made by these hybridomas for various scientific investigations.
Endothelial cells, which form a continuous lining of the vascular systems, perform a variety of functions, including control of coagulation, regulation of platelet function and thrombosis and transport of cells and metabolic components to and from circulation. Much evidence supports the role of vascular endothelial cells in the initiation of inflammatory and immunological responses, including adhesion to circulating leukocytes, stimulation of intravascular coagulation, and presentation of antigen to T lymphocytes.
The rheumatoid (RA) synovium contains numerous new and developing blood vessels, perhaps fueled by angiogenic factors released by macrophages within these tissues. The inflamed endothelium may have different phenotypic and functional characteristics from that found in other noninflamed tissues. For instance, there may be a distinct leukocyte-endothelium recognition system that directs the extravasation of leukocytes into inflamed synovium.
While markers such as monoclonal antibodies (mAbs) BW200, PAL-E, and E431 are pan-endothelial, recent evidence has indicated that vascular endothelial displays phenotypic heterogeneity, as determined by monoclonal antibodies detecting subsets of endothelial cells (Alles, J. U., and K. Bosslet. 1986. Immunohistochemical and immunochemical characterization of a new endothelial cell-specific antigen. J. Histochem. Cytochem. 34:209-214; Hagemeier, H-H., E. Vollmer, S. Goerdt, K. Schulze-Osthoff, and C. Sorg. 1986. Monoclonal antibody reacting with endothelial cells and budding vessels in tumors and inflammatory tissues, and non-reactive with normal adult tissues. Int. J. Cancer 38:481-488; Schlingemann, R. O., G. M. Dingjan, J. J. Emeis, J. Blok, S. O. Warnaar, and D. J. Ruiter. 1985. Monoclonal antibody PAL-E specific for endothelium. Lab. Invest. 52:71-76; Scully, P. A. H. K. Steinman, C. Kennedy, K. Trueblood, D. M. Frisman, and J. R. Voland. 1988. AIDS-related Kaposi's sarcoma displays differential expression of endothelial surface antigens. Am. J. Pathol. 130:244-251). A monoclonal antibody has been described which reacts preferentially with endothelial cells of capillaries, small and medium sized veins, and venules in normal and tumor tissues (Schlingemann, R. O., G. M. Dingjan, J. J. Emeis, J. Blok, S. O. Warnaar, and D. J. Ruiter. 1985. Monoclonal antibody PAL-E specific for endothelial. Lab. Invest. 52:71-76). Monoclonal antibody EN 7/44 reacts with endothelial cells of budding vessels in human tumors, inflammatory tissues, and placentas, as well as with cultured human umbilical vein endothelial cells (HUVECS), but does not react with endothelial cells of normal tissues (Hagemeier, H-H., E. Vollmer, S. Goerdt, K. Schulze-Osthoff, and C. Sorg. 1986. Monoclonal antibody reacting with endothelial cells and budding vessels in tumors and inflammatory tissues, and non-reactive with normal adult tissues. Int. J. Cancer 38:481-488 ). Monoclonal antibody 1F10 recognizes an endothelial cell surface antigen abundantly expressed in continuous endothelia, but rarely expressed in liver sinusoidal endothelia (Goerdt, S., G. Zwaldo, R. Schlegel, H.-H. Hagemeier, and C. Sorg. 1987. Characterization and expression kinetics of an endothelial cell activation antigen present in vivo only in acute inflammatory tissue. Exp. Cell. Biol. 55:117-126). In contrast monoclonal antibody 4A11, described here, appears to detect an antigen distinct from those antigens recognized by other monoclonal antibodies, since it does not react with cultured human umbilical vein endothelial cells and is selective for endothelial in synovium, lymphoid tissues, and malignant or inflamed tissues.
Recently, antigens have been detected on endothelial cells which have been activated with stimuli such as cytokines (Munro, J. M., J. S. Pober, and R. S. Cotran. 1989. Tumor necrosis factor and interferon gamma induce distinct patterns of endothelial activation and associated leukocyte accumulation in skin of Papio anubis. Am. J. Pathol. 135:121-133; Bevilacqua, M. P., J. S. Pober, D. L. Mendrick, R. S. Cotran, and M. A. Gimbrone. 1987. Identification of an inducible endothelial-leukocyte adhesion molecule. Proc. Natl. Acad. Sci. USA 84:9238-9342). Monoclonal antibody 4D10 reacts with lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF), interleukin-1 (IL-1) and phorbol myristate acetate (PMA) stimulated cultured human umbilical vein endothelial cells in culture and with endothelial cells in tissues in acute inflammatory conditions, such as atopic dermatitis (Goerdt, S., G. Zwaldo, R. Schlegel, H.-H. Hagemeier, and C. Sorg. 1987. Characterization and expression kinetics of an endothelial cell activation antigen present in vivo only in acute inflammatory tissue. Exp. Cell. Biol. 55:117-126). 4D10 antigen is not present in rheumatoid arthritis (RA) synovial tissues. Antigens such as ELAM-1 are expressed selectively on cultured human umbilical vein endothelial cells stimulated with interlukin-1 or tumor necrosis factor-alpha, but not on resting cultured human umbilical vein endothelial cells (Bevilacqua, M. P., J. S. Pober, D. L. Mendrick, R. S. Cotran, and M. A. Gimbrone. 1987. Identification of an inducible endothelial-leukocyte adhesion molecule. Proc. Natl. Acad. Sci. USA 84:9238-9342; Cotran, R. S., M. A. Gimbrone jr., M. P. Bevilacqua, D. L. Mendrick, and J. S. Pober. 1986. Induction and detection of a human endothelial activation antigen in vivo. J. Exp. Med. 164:661-666; Pober, J. S., and M. A. Gimbrone 1982. Expression of Ia-like antigens by human vascular endothelial cells is inducible in vitro: Demonstration by monoclonal antibody binding and immunoprecipitation. Proc. Natl. Acad. Sci 79:6641-6645; Bevilacqua, M. P., J. S. Pober, M. E. Wheeler, R. S. Cotran, and M. A. Gimbrone Jr. 1985. Interleukin-1 activation of vascular endothelium. Effects on procoagulant activity and leucocyte adhesion. Am. J. Pathol. 121:394-403; Bevilacqua, M. P., S. Stentgelin, M. A. Gimbrone Jr., and B. Seed. 1989. Endothelial leukocyte adhesion molecule 1: an inducible receptor for neutrophils related to complement regulatory proteins and lectins. Science 243:160-1165; Pober, J. S., D. L. Bevilacqua, L. A. Mendrick, W. Fiers, and M. A. Gimbrone Jr. 1986. Two distinct monokines, interleukin 1 and tumor necrosis factor, each independently induce biosynthesis and transient expression of the same antigen on the surface of cultured human vascular endothelial cells. J. Immunol. 136:1680-1687). Endothelial leukocyte adhesion molecule-1 (ELAM-1) is expressed on endothelium in delayed hypersensitivity reactions and on endothelium of inflamed but not normal tissues and in part mediates monocyte adherence to endothelial cells (Cotran, R. S., M. A. Gimbrone jr., M. P. Bevilacqua, D. L. Mendrick, and J. S. Pober. 1986. Induction and detection of a human endothelial activation antigen in vivo. J. Exp. Med. 164:661-666; Carlos, T., and J. M. Harlan. 1990. Membrane proteins involved in phagocyte adherence to endothelium. Immunological Reviews 114:1-28). In contrast, monoclonal antibody 4A11 does not detect an antigen upregulated by stimulation of cultured human umbilical vein endothelial cells with the cytokines examined.
Endothelial cells are strategically located at the interface between the circulation and the extravascular tissues and perform many functions, including control of coagulation and regulation of thrombus formation (Duijvestijn, A. M., M. Kerkhove, R. F. Bargatze, and E. C. Butcher, 1987. Lymphoid tissue and inflammation-specific endothelial cell differentiation defined by monoclonal antibodies. J. Immuno. 138:713-179). One particularly important function of endothelium is its role in mediating leukocyte extravasation from blood into sites of inflammation (Duijvestijn, A. M., M. Kerkhove, R. F. Bargatze, and E. C. Butcher, 1987. Lymphoid tissue and inflammation-specific endothelial cell differentiation defined by monoclonal antibodies. J. Immunol. 138:713-179). For instance, high endothelial venules in mucosal lymphoid organs in nonmucosal lymph nodes, and inflamed synovium, express organ-specific ligands recognized by complementary homing receptor molecules on circulating lymphocytes (Duijvestijn, A. M., M. Kerkhove, R. F. Bargatze, and E. C. Butcher, 1987. Lymphoid tissue and inflammation-specific endothelial cell differentiation defined by monoclonal antibodies. J. Immunol. 138:713-179; Butcher, E. C., R. G. Scollay, and I. L. Weissman. 1980. Organ specific lymphocyte migration: Mediation by highly selective lymphocyte interactions with organ-specific determinants on high endothelial venules. Eur. J. Immunol. 10:556-561; Chin, Y. H., R. A. Rasmussen, J. J. Woodruff, and T. G. Easton. 1986. A monoclonal anti-HEBFpp antibody with specificity for lymphocyte surface molecules mediating adhesion to Peyer's patch high endothelium of the rat. J. Immunol. 136:2556-2561; Gallatin, W. M., E. C. Butcher, and I. L. Weissman. 1983. A cell surface molecule involved in organ-specific homing of lymphocytes. Nature 304:30-34; Jalkanen, S. T., R. F. Bargatze, L. R. Herron, E. C. Butcher. 1986 A lymphoid cell surface glycoprotein involved in endothelial cell recognition and lymphocyte homing in man. Eur. J. Immunol. 16:1195-1202; Rasmussen, R. A., Y. H. Chin, J. J. Woodruff, and T. G. Easton. 1985. Lymphocyte recognition of lymph node high endothelium. VII. Cell surface proteins involved in adhesion defined by monoclonal anti-HEBF.sub.LN (A.11) antibody. J. Immunol. 135:19-24). Unlike the synovial reactivity of HECA-452, which has been reported by various authors either not to be present in synovial tissue from rheumatoid arthritis patients or to react with both high endothelial venules and synovial dendritic cells, the inventor does not find 4A11 reactivity on rheumatoid arthritis synovial dendritic cells (Jalkanen, S. 1989. Leukocyte-endothelial cell interaction and the control of leukocyte migration into inflamed synovium. Springer Semin. Immunopathol. 11:187-198; van Dinther-Janssen, A. C. H. M., S. T. Pals, R. Scheper, F. Breedveld, and C. J. L. M. Meijer. 1990. Dendritic cells and high endothelial venules in rheumatoid synovial membranes).